Progress 11/15/03 to 11/14/04
Outputs
To modify the benzenoid pathway in petunia we have used two different approaches. 1. To shift the metabolic flux to benzyl alcohol and thus to increase its amount, the expression of the benzoyl-coenzyme A:benzyl alcohol/phenylethanol benzoyl transferase (BPBT) was down-regulated using RNA interference-based (RNAi) suppression. The RNAi construct for BPBT was generated containing two copies of a 320-bp fragment from the 5' end of the BPBT coding region in an inverted-repeat orientation under the control of the petal-specific LIS promoter. The binary vector with LIS promoter was developed based on pCAMBIA1303 binary vector, in which CaMV 35S promoter, gusA, and mGFP were removed, and a 1 kb region of the LIS promoter was inserted between EcoRI and SacI restriction sites of the polylinker region of the vector. Petunia hybrida cv Mitchell leaf disks were transformed using Agrobacterium tumefaciens (GV 3101) containing the recombinant plasmids. Explants transformed with the
modified pCAMBIA1303 binary vector were grown on hygromycin selection medium (10 mg/L). 30 individual lines were generated which are currently blooming in the greenhouse at Purdue University. Analysis of down-regulation of BPBT expression in petunia flowers was confirmed by Northern blot analysis, which revealed that only in 10 individual lines BPBT expression was completely eliminated while in others it was decreased by 2-fold. Analysis of scent emission showed that lines with complete suppression of BPBT expression do not emit benzylbenzoate nor phenylethylbenzoate, which are the products of BPBT enzyme. Flowers from transgenic plants with complete and moderate reduction in benzylbenzoate and phenylethylbenzoate emission were used for metabolic profiling of volatile compounds and intracellular intermediates and end products. For metabolic flux analysis petal tissue will be supplied with deuterium labeled Phe and the labeling kinetics of the endogenous pools of
phenylpropanoid/benzenoid metabolites and the volatiles emitted to the gas phase will be analyzed at different time points over a 4-hour period. The obtained results will be incorporated in a flux model. These experiments are in progress now. Also several transgenic plants with complete and moderate reduction in benzylbenzoate and phenylethylbenzoate emission were self-pollinated to produce T1 plant populations. 2. To increase the amount of emitted benzylacetate we proposed to overexpress Clarkia breweri acetyl-coenzyme A:benzyl alcohol acetyltransferase (BEAT) which catalyzes the formation of benzylacetate from benzylalcohol and acetyl-CoA. Clarkia breweri BEAT cDNA, 1.3 kb in size, was inserted in the sense orientation between the petal-specific Lis promoter and the 3' terminator sequence of the NOS gene of the modified binary vectors pCAMBIA1303 and pMDC100, described above, and introduced into petunia plants by Agrobacterium tumefaciens-mediated transformation. These experiments
are in progress now.
Impacts
Floral scent is an important character for both crop plants and ornamentals. Floral scent in insect-pollinated, agronomically important plants is crucial for seed and fruit set. For ornamental plants, scent increases the plant's aesthetic value, thus directly benefiting consumers. Recent advances in the isolation of scent biosynthetic genes have opened up the possibility for the genetic manipulation of floral scent, which was only partially successful. The long-term goal of this research is to use tools of metabolic engineering in combination with metabolic flux analysis and modeling to improve and enhance floral scent in ornamental and cut flowers. The research is expected to provide a quantitative description of the metabolic networks involved in scent production in petunia and carnation, and uncover the effects of targeted metabolic engineering on steady-state levels of metabolites and fluxes in the pathways. This research will also build a foundation for the future
metabolic engineering of floral scent of crop plants cultivated for their seed, forage, or food value, with the purpose of increasing their attractiveness to pollinators and thus ensuring efficient pollination.
Publications
- Dudareva N, Pichersky E. 2005. Metabolic engineering of floral scent of ornamentals. Journal of Crop Improvement, (in press).
- Dudareva N, Negre F. 2005. Practical applications of research into the regulation of plant volatile emission. Current Opinion in Plant Biology, 8: 113-118.
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Progress 10/01/03 to 09/29/04
Outputs
To modify the benzenoid pathway in petunia we have used two different approaches. 1. To shift the metabolic flux to benzyl alcohol and thus to increase its amount, the expression of the benzoyl-coenzyme A:benzyl alcohol/phenylethanol benzoyl transferase (BPBT) was down-regulated using RNA interference-based (RNAi) suppression. Using a functional genomic approach we have isolated BPBT from Petunia hybrida cv. 'Mitchell' and found that the enzyme can use both benzyl alcohol and phenylethanol as substrates in vivo with slight favor toward benzyl alcohol. Two types of RNAi constructs for BPBT were made, one under the control of the 35S promoter and the other under the control of the petal-specific LIS promoter. The binary vector with LIS promoter was developed based on pCAMBIA1303 binary vector, in which CaMV 35S promoter, gusA, and mGFP were removed, and a 1 kb region of the LIS promoter was inserted between EcoRI and SacI restriction sites of the polylinker region of
the vector. Petunia leaf disks were transformed using Agrobacterium tumefaciens (GV 3101) containing the recombinant plasmids. Explants transformed with the modified pCAMBIA1303 binary vector were grown on hygromycin selection medium. In order to be able to make crosses between transgenic lines in the future, similar set of constructs were prepared based on pMDC100 binary vector with kanamycin resistance (NPT II) using the Gateway technology and used for petunia transformation. These experiments are in progress now. 2. To increase the amount of emitted benzylacetate we proposed to overexpress Clarkia breweri acetyl-coenzyme A:benzyl alcohol acetyltransferase (BEAT) which catalyzes the formation of benzylacetate from benzylalcohol and acetyl-CoA. Clarkia breweri BEAT cDNA, 1.3 kb in size, was inserted in the sense orientation between the petal-specific Lis promoter and the 3' terminator sequence of the NOS gene of the modified binary vectors pCAMBIA1303 and pMDC100, described above,
and introduced into petunia plants by Agrobacterium tumefaciens-mediated transformation. These experiments are in progress now. To modify the terpenoid pathway in chrysanthemum we have also prepared two types of construct. To increase the supply of FPP, which is the precursor of sesquiterpenes, the Arabidopsis FPP synthase, 1 kb in size, was inserted in the sense orientation between the petal-specific Lis promoter and the 3' terminator sequence of the NOS gene of the modified binary vector pMDC100 and introduced into chrysanthemum plants by Agrobacterium tumefaciens-mediated transformation. To increase the supply of GPP, which is the precursor of monoterpenes, the snapdragon gene encoding the small subunit of GPP synthase, 0.9 kb in size, was inserted in the sense orientation between the petal-specific Lis promoter and the 3' terminator sequence of the NOS gene of the modified binary vectors pCAMBIA1303 and pCGN1559, and introduced into snapdragon and chrysanthemum plants by
Agrobacterium tumefaciens-mediated transformation.
Impacts
Results from this research will provide the knowledge base for manipulation of the output of volatile compounds by recombinant DNA technologies. New crops with modified composition of volatiles and newly introduced aroma would benefit US agriculture by increasing crop productivity, pest resistance, and the value of ornamentals.
Publications
- Dudareva N, Pichersky E, Gershenzon J. 2004. Biochemistry of plant volatiles. Plant Physiology, 135: 1893-1902.
- Dudareva N. 2003. Floral scent. In R.M. Goodman (ed), Encyclopedia of Plant and Crop Science, Marcel Dekker, Inc Agropedia.
- Rhodes D, Peel GJ, Dudareva N. 2003. Engineering pathways of secondary metabolism. In R.M. Goodman (ed), Encyclopedia of Plant and Crop Science, Marcel Dekker, Inc Agropedia.
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